Volume 12, Issue 21, Pages 1877-1884 (October 2002) Genetically Expressed Cameleon in Drosophila melanogaster Is Used to Visualize Olfactory Information in Projection Neurons  André Fiala, Thomas Spall, Sören Diegelmann, Beate Eisermann, Silke Sachse, Jean-Marc Devaud, Erich Buchner, C.Giovanni Galizia  Current Biology  Volume 12, Issue 21, Pages 1877-1884 (October 2002) DOI: 10.1016/S0960-9822(02)01239-3

Figure 1 Cameleon Expression and Imaging Setup (A) Whole-mount preparation of a Drosophila brain expressing cameleon 2.1 in projection neurons under the control of the Gal4 line GH 146. The image was taken with a color CCD camera (Axiocam, Zeiss) and was contrast enhanced with an unsharp mask filter (PhotoShop 5.5 program, Adobe). AL: antennal lobe, CX: calyx, LPL: lateral protocerebral lobe, arrowheads: inner antennocerebral tract. The scale bar represents 50 μm. Approximate areas imaged for the AL (see Figure 2) and the CX (see Figure 3) are indicated by boxes. (B) Imaging setup for in vivo cameleon imaging. The 440 nm excitation is provided by a xenon lamp and a monochromator and is focused via a water immersion objective onto the brain. Emission wavelengths passing a 470 nm dichroic long pass mirror (DCLP 470 nm) are split up by a second dichroic long pass mirror (DCLP 520 nm), and the two beams are guided through band pass filters (BP 473–494 nm for the ECFP emission, and BP 530–565 nm for the EYFP emission). The two emissions are simultaneously recorded by two independent CCD cameras. Current Biology 2002 12, 1877-1884DOI: (10.1016/S0960-9822(02)01239-3)

Figure 2 Odorant-Evoked Calcium Responses in the Drosophila Antennal Lobe (A) Frontal view of the two ALs of one brain, as seen with EYFP fluorescence, after contrast enhancement. Compare this figure with Figure 1A for orientation and position of the area in the whole brain. The squares mark the positions evaluated in (C). This animal's position was slightly oblique; therefore, the symmetry line is not vertical. The scale bar represents 25 μm. (B) Comparison of spatial FRET difference patterns between animals. Color-coded results from three different individuals for single stimulations per odorant for three different odorants are shown. To the right, areas with the strongest activity for each odorant are superimposed onto an EYFP fluorescence image providing a morphological view. Here, purple areas indicate responses to octanol (OCT), cyan areas indicate responses to benzaldehyde (BEA), and yellow areas indicate responses to isoamyl acetate (IAA). Areas responding both to BEA and IAA are green, and overlapping regions of OCT and BEA are blue. While the glomeruli could not unequivocally be identified from their morphology, the similarity of the activity patterns between animals suggests that homologous glomeruli are excited by the same odorant in different animals. In each image, false-color coding scales the responses between −2.0% EYFP/ECFP and the maximum, which is indicated in each image (e.g., 5.8 for the upper octanol). (C) Time courses of the responses to the odorants BEA, IAA, OCT, and the control mineral oil (MOL) in fly 2 (single stimulation each). Two different glomeruli are evaluated; the upper row of graphs correspond to position 1 in (A), and the lower row of graphs correspond to position 2 in (A). For each glomerulus and odorant, the response is shown for the EYFP (yellow line) and the ECFP (blue line) signal (both as % ΔF/F). The EYFP/ECFP ratio change is shown as a black line (also as % ratio). All curves are shifted to baseline before odorant stimulus onset (t = 0 s). Odorant stimulation is indicated by the gray bar. (D) Spatiotemporal response patterns, shown as a sequence of images from the AL. Adjacent images are 0.66 s apart, i.e., every second frame is shown. Frame numbers are indicated in the upper-right corner of each frame, with 0 denoting stimulus onset. Odorant stimulus is indicated by the red bar. Ratio values above 0 are false-color coded on top of the morphological view of the two ALs. Note the bilateral symmetry of the odorant-evoked signals, the spatial differences between OCT and IAA, and the very weak responses to BEA in this animal. See also the movies in the Supplementary Material available with this article online. Current Biology 2002 12, 1877-1884DOI: (10.1016/S0960-9822(02)01239-3)

Figure 3 Odorant-Evoked Calcium Responses in the Drosophila Calyx (A) A view onto one calyx, as seen with EYFP fluorescence and after contrast-enhancement, with the adjacent inner antennocerebral tract. The numbered squares mark the positions evaluated in (C). Compare this figure with Figure 1A for position of the area in the whole brain. The scale bar represents 25 μm. (B) Comparison of spatial FRET difference patterns for repeated stimulation within one animal, with a single stimulation in each trial. Color-coded results for the three different odorants benzaldehyde (BEA), isoamyl acetate (IAA), and octanol (OCT) are shown. In each image, false-color coding scales the responses between −2.0 and the maximum, which is indicated in each image (e.g., 2.6 for the upper octanol). The outline of the CX is indicated by a black line. (C) Time courses of the responses to single stimulations with BEA, OCT, IAA, and the control mineral oil (MOL). Two different coordinates are evaluated: coordinate 1 in (A) refers to the upper row, and coordinate 2 in (A) refers to the lower row. For each coordinate, the response is shown for the EYFP (yellow line) and the ECFP (blue line) signal (both as % ΔF/F). The EYFP/ECFP ratio is shown as a black line (also as % ratio). All curves are shifted to baseline before odorant stimulus onset (t = 0 s). Odorant stimulation is indicated by the gray bar. (D) Averages of the time courses of three responses to stimulations with BEA, OCT, IAA, and MOL. The same animal and coordinates that are shown in (C) are shown. Current Biology 2002 12, 1877-1884DOI: (10.1016/S0960-9822(02)01239-3)

Figure 4 Spatiotemporal Response Patterns in the Calyx Odorant-evoked response patterns, shown as a sequence of images from the calyx. Adjacent images are 0.66 s apart. The odorant stimulus is indicated by the red bar. Ratio values above 0 are false-color coded on top of the morphological view of the calyx. Note the spatial differences between BEA, OCT, and IAA. See also the movies in the Supplementary Material available with this article online. The scale bar represents 25 μm. Current Biology 2002 12, 1877-1884DOI: (10.1016/S0960-9822(02)01239-3)